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Epidermal growth factor receptor phosphorylation sites Ser991 and Tyr998 are implicated in the regulation of receptor endocytosis and phosphorylations at Ser1039 and Thr1041.

Tong J, Taylor P, Peterman SM, Prakash A, Moran MF - Mol. Cell Proteomics (2009)

Bottom Line: Compared with wild type EGFR the Y998F variant had diminished EGF-stimulated interaction with the ubiquitin E3 ligase CBL, and the S991A variant had decreased associated ubiquitin.The endocytosis-defective mutant receptors were found to have elevated phosphorylation at positions Ser(1039) and Thr(1041).These results suggest that coordinated phosphorylation of EGFR involving sites Tyr(998), Ser(991), Ser(1039), and Thr(1041) governs the trafficking of EGF receptors.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular Structure and Function, The Hospital For Sick Children, and The McLaughlin Centre For Molecular Medicine, Toronto, Ontario M5G 1L7, Canada.

ABSTRACT
Aberrant expression, activation, and down-regulation of the epidermal growth factor receptor (EGFR) have causal roles in many human cancers, and post-translational modifications including phosphorylation and ubiquitination and protein-protein interactions directly modulate EGFR function. Quantitative mass spectrometric analyses including selected reaction monitoring (also known as multiple reaction monitoring) were applied to the EGFR and associated proteins. In response to epidermal growth factor (EGF) stimulation of cells, phosphorylations at EGFR Ser(991) and Tyr(998) accumulated more slowly than at receptor sites involved in RAS-ERK signaling. Phosphorylation-deficient mutant receptors S991A and Y998F activated ERK in response to EGF but were impaired for receptor endocytosis. Consistent with these results, the mutant receptors retained a network of interactions with known signaling proteins including EGF-stimulated binding to the adaptor GRB2. Compared with wild type EGFR the Y998F variant had diminished EGF-stimulated interaction with the ubiquitin E3 ligase CBL, and the S991A variant had decreased associated ubiquitin. The endocytosis-defective mutant receptors were found to have elevated phosphorylation at positions Ser(1039) and Thr(1041). These residues reside in a serine/threonine-rich region of the receptor previously implicated in p38 mitogen-activated protein kinase-dependent stress/cytokine-induced EGFR internalization and recycling (Zwang, Y., and Yarden, Y. (2006) p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy. EMBO J. 25, 4195-4206). EGF-induced phosphorylations at Ser(1039) and Thr(1041) were blocked by treatment of cells with SB-202190, a selective inhibitor of p38. These results suggest that coordinated phosphorylation of EGFR involving sites Tyr(998), Ser(991), Ser(1039), and Thr(1041) governs the trafficking of EGF receptors. This reinforces the notion that EGFR function is manifest through spatially and temporally controlled protein-protein interactions and phosphorylations.

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Spatial and temporal dynamics of wild type EGFR and phosphorylation-deficient variants. HEK293-based cells stably expressing WT, S991A, and Y998F EGFR proteins (as indicated) fused to GFP were treated with EGF (100 ng/ml) for 0, 5, and 15 min at 37 °C as indicated, then fixed, and localized by using fluorescence microscopy. Long arrows indicate staining at the cell periphery near plasma membranes, and the short arrows indicate regions of aggregated intracellular staining that may correspond to the endosomal compartment. Scale bar, 100 μm.
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Figure 3: Spatial and temporal dynamics of wild type EGFR and phosphorylation-deficient variants. HEK293-based cells stably expressing WT, S991A, and Y998F EGFR proteins (as indicated) fused to GFP were treated with EGF (100 ng/ml) for 0, 5, and 15 min at 37 °C as indicated, then fixed, and localized by using fluorescence microscopy. Long arrows indicate staining at the cell periphery near plasma membranes, and the short arrows indicate regions of aggregated intracellular staining that may correspond to the endosomal compartment. Scale bar, 100 μm.

Mentions: To examine endocytosis of WT EGFR and the phosphorylation-defective receptor variants, localization of chimeric receptors containing carboxyl-terminal green fluorescent protein (GFP) was monitored by fluorescence microscopy. In serum-starved HEK-EGFR, HEK-S991A, and HEK-Y998F cells, the receptors were concentrated near the cell periphery consistent with plasma membrane localization (Fig. 3, left panels, long arrows). After exposure to 100 ng/ml EGF and incubation at 37 °C for 5 and 15 min, WT EGFR staining at the cell margins was diminished and became internalized and concentrated into intracellular aggregates characteristic of endosomes (Fig. 3, upper row, short arrows). By contrast, Y998F and S991A mutant receptors displayed far less internalization/aggregation and remained largely concentrated at the cell periphery (Fig. 3, middle and lower rows, long arrows). These observations demonstrate defective endocytosis of the mutant receptors. The endocytosis defect of EGFR Y998F is expected based on Sorkin et al. (16). The cell staining was categorized into two types: WT-like, typified by internalized and aggregated receptors and little or no staining at the cell margins, and mutant-like, which lacked intracellular aggregates and retained concentrated staining at the cell margins. After tabulating these patterns in 100 cells in three separate experiments, ∼60% of WT receptors displayed the WT-like pattern of internalized receptors (diminished peripheral staining accompanied by internalized aggregates), whereas only ∼30% of Y998F and 25% of S991A receptors showed this pattern.


Epidermal growth factor receptor phosphorylation sites Ser991 and Tyr998 are implicated in the regulation of receptor endocytosis and phosphorylations at Ser1039 and Thr1041.

Tong J, Taylor P, Peterman SM, Prakash A, Moran MF - Mol. Cell Proteomics (2009)

Spatial and temporal dynamics of wild type EGFR and phosphorylation-deficient variants. HEK293-based cells stably expressing WT, S991A, and Y998F EGFR proteins (as indicated) fused to GFP were treated with EGF (100 ng/ml) for 0, 5, and 15 min at 37 °C as indicated, then fixed, and localized by using fluorescence microscopy. Long arrows indicate staining at the cell periphery near plasma membranes, and the short arrows indicate regions of aggregated intracellular staining that may correspond to the endosomal compartment. Scale bar, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2742444&req=5

Figure 3: Spatial and temporal dynamics of wild type EGFR and phosphorylation-deficient variants. HEK293-based cells stably expressing WT, S991A, and Y998F EGFR proteins (as indicated) fused to GFP were treated with EGF (100 ng/ml) for 0, 5, and 15 min at 37 °C as indicated, then fixed, and localized by using fluorescence microscopy. Long arrows indicate staining at the cell periphery near plasma membranes, and the short arrows indicate regions of aggregated intracellular staining that may correspond to the endosomal compartment. Scale bar, 100 μm.
Mentions: To examine endocytosis of WT EGFR and the phosphorylation-defective receptor variants, localization of chimeric receptors containing carboxyl-terminal green fluorescent protein (GFP) was monitored by fluorescence microscopy. In serum-starved HEK-EGFR, HEK-S991A, and HEK-Y998F cells, the receptors were concentrated near the cell periphery consistent with plasma membrane localization (Fig. 3, left panels, long arrows). After exposure to 100 ng/ml EGF and incubation at 37 °C for 5 and 15 min, WT EGFR staining at the cell margins was diminished and became internalized and concentrated into intracellular aggregates characteristic of endosomes (Fig. 3, upper row, short arrows). By contrast, Y998F and S991A mutant receptors displayed far less internalization/aggregation and remained largely concentrated at the cell periphery (Fig. 3, middle and lower rows, long arrows). These observations demonstrate defective endocytosis of the mutant receptors. The endocytosis defect of EGFR Y998F is expected based on Sorkin et al. (16). The cell staining was categorized into two types: WT-like, typified by internalized and aggregated receptors and little or no staining at the cell margins, and mutant-like, which lacked intracellular aggregates and retained concentrated staining at the cell margins. After tabulating these patterns in 100 cells in three separate experiments, ∼60% of WT receptors displayed the WT-like pattern of internalized receptors (diminished peripheral staining accompanied by internalized aggregates), whereas only ∼30% of Y998F and 25% of S991A receptors showed this pattern.

Bottom Line: Compared with wild type EGFR the Y998F variant had diminished EGF-stimulated interaction with the ubiquitin E3 ligase CBL, and the S991A variant had decreased associated ubiquitin.The endocytosis-defective mutant receptors were found to have elevated phosphorylation at positions Ser(1039) and Thr(1041).These results suggest that coordinated phosphorylation of EGFR involving sites Tyr(998), Ser(991), Ser(1039), and Thr(1041) governs the trafficking of EGF receptors.

View Article: PubMed Central - PubMed

Affiliation: Program in Molecular Structure and Function, The Hospital For Sick Children, and The McLaughlin Centre For Molecular Medicine, Toronto, Ontario M5G 1L7, Canada.

ABSTRACT
Aberrant expression, activation, and down-regulation of the epidermal growth factor receptor (EGFR) have causal roles in many human cancers, and post-translational modifications including phosphorylation and ubiquitination and protein-protein interactions directly modulate EGFR function. Quantitative mass spectrometric analyses including selected reaction monitoring (also known as multiple reaction monitoring) were applied to the EGFR and associated proteins. In response to epidermal growth factor (EGF) stimulation of cells, phosphorylations at EGFR Ser(991) and Tyr(998) accumulated more slowly than at receptor sites involved in RAS-ERK signaling. Phosphorylation-deficient mutant receptors S991A and Y998F activated ERK in response to EGF but were impaired for receptor endocytosis. Consistent with these results, the mutant receptors retained a network of interactions with known signaling proteins including EGF-stimulated binding to the adaptor GRB2. Compared with wild type EGFR the Y998F variant had diminished EGF-stimulated interaction with the ubiquitin E3 ligase CBL, and the S991A variant had decreased associated ubiquitin. The endocytosis-defective mutant receptors were found to have elevated phosphorylation at positions Ser(1039) and Thr(1041). These residues reside in a serine/threonine-rich region of the receptor previously implicated in p38 mitogen-activated protein kinase-dependent stress/cytokine-induced EGFR internalization and recycling (Zwang, Y., and Yarden, Y. (2006) p38 MAP kinase mediates stress-induced internalization of EGFR: implications for cancer chemotherapy. EMBO J. 25, 4195-4206). EGF-induced phosphorylations at Ser(1039) and Thr(1041) were blocked by treatment of cells with SB-202190, a selective inhibitor of p38. These results suggest that coordinated phosphorylation of EGFR involving sites Tyr(998), Ser(991), Ser(1039), and Thr(1041) governs the trafficking of EGF receptors. This reinforces the notion that EGFR function is manifest through spatially and temporally controlled protein-protein interactions and phosphorylations.

Show MeSH
Related in: MedlinePlus